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Expression of hBMP4 and hBMP7 in Chinese Hamster Ovary Cells

Author: DengChunMei
Tutor: ZhangYanDing
School: Fujian Normal University
Course: Developmental Biology
Keywords: rhBMP4 rhBMP7 CHO cells exogenous protein expression
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 4
Quote: 0
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Abstract


Bone Morphogenetic Proteins are growth factors, members of the transforming growth factorβsuperfamily, which main active form are homologous or glycosylated heterodimer. They can make undifferentiated mesenchymal cells differentiate into osteoblasts and form bone tissue. Many BMPs have osteoinductive activity, BMP4 and BMP7 have the strongest induced activity among them. Later research found that BMPs not only have osteoinductive activity, but also can regulate the cell growth, proliferation and differentiation. At the same time, BMPs play an important role in determination and differentiation of various tissues and organs, for example the brain, kidney, liver, urinary system and so on.Scholars in the country mainly use E. coli or pichia pastoris express rhBMPs. These expression system cost is low and operation is very simple, but the structure of the product is different from the natural protein. So in this experiment, we try to use Chinese hamster ovary cells express hBMP4 and hBMP7. Our work include:①Firstly construct the hBMP4 and hBMP7 recombinant expression vector, and then electrictransfected into the CHO/DG44 cells. Finally identify the gene integration and transcription by genomic PCR and RT-PCR.②After a preliminary purification of the expressed product, WESTERN-BLOT analysis showed that the existence of rhBMP4 dimer and monomer.③The expression level of rhBMP4 increased 2-fold by methotrexate pressure, the result showed rhBMP4 expression quantity was 330ng/ml.④High expression cell lines have been selected by the limited dilution method, the expression of rhBMP4 reached 583ng/ml.⑤We detected the biological activity of rhBMP4 in vitro, the results showed that rhBMP4 can increase the alkaline phosphatase activity of NIH3T3 cells.⑥DOT-BLOT and ELISA analysis showed that the CHO/DG44 cells contain hBMP7 recombinant plasmid can express rhBMP7.

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